Liposomes are microscopic particles that are made up of one or more lipid bilayers enclosing an internal compartment. Liposomes can be categorized into multilamellar vesicles, multivesicular liposomes, unilamellar vesicles and giant liposomes. Multilamellar liposomes (also known as multilamellar vesicles or “MLV”) contain multiple concentric bilayers within each liposome particle, resembling the “layers of an onion”. Multivesicular liposomes consist of lipid membranes enclosing multiple non-concentric aqueous chambers. Liposomes that enclose a single internal aqueous compartment include small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs). LUVs and SUVs range in size from about 50 to 500 nm and 20 to 50 nm respectively. Giant liposomes typically range in size from 5000 nm to 50,000 nm and are used mainly for studying mechanochemical and interactive features of lipid bilayer vesicles in vitro (Needham et al., Colloids and Surfaces B: Biointerfaces (2000) 18: 183-195).
Liposomes have been widely studied and used as carriers for a variety of agents such as drugs, cosmetics, diagnostic reagents, and genetic material. Since liposomes consist of non-toxic lipids, they generally have low toxicity and therefore are useful in a variety of pharmaceutical applications. In particular, liposomes are useful for increasing the circulation lifetime of agents that have a short half-life in the bloodstream. Liposome-encapsulated drugs often have biodistributions and toxicities which differ greatly from those of free drug. For specific in vivo delivery, the sizes, charges and surface properties of these carriers can be changed by varying the preparation methods and by tailoring the lipid makeup of the carrier. For instance, liposomes may be made to release a drug more quickly by decreasing the acyl chain length of a lipid making up the carrier.
Liposomes containing metal ions encapsulated in the interior of the vesicle have been used in diagnostic applications. For example, liposomes have been used for delivery of contrast agents with the goal of accumulating a contrast agent at a desired site within the body of a subject. In the latter application, liposomes have mainly been used for delivery of diagnostic radionucleotides and paramagnetic metal ions in gamma and magnetic resonance imaging, respectively. This includes liposomal encapsulation of radionucleotides such as 111In, 99mTc and 67Ga and paramagnetic ions such as Gd, Mn and manganese oxide. Two methods are typically employed to prepare liposomes for imaging purposes. In the first method, the metal is converted to a soluble chelate and then introduced into the aqueous interior of a liposome. In the second method, a chelating agent derivatized with a lipophilic group is anchored to the liposome surface during or after liposome preparation.
Manganese and non-transition metal ions have also been involved in methods for encapsulation of ionizable agents into liposomes containing an ionophore inserted in the liposome membrane (see U.S. Pat. No. 5,837,282 and Fenske et al., Biochim. Biophys. Acta (1998) 1414: 188-204). In this method, the ionophore translocates the metal ion across the liposome membrane in exchange for protons, thereby establishing a pH gradient. The establishment of an appropriate pH gradient across the liposome bilayer allows the ionizable agent to be encapsulated since the agent can readily cross the liposomal bilayer in the neutral form and subsequently become encapsulated and trapped within the aqueous interior of the liposome due to conversion to the charged form (see Mayer et al., U.S. Pat. Nos. 6,083,530, 5,616,341, 5,795,589 and 5744158; Mayer et al., Biochimica et Biophysica Acta (1986) 857:123). This work arose from mechanistic studies completed by Deamer et al., (Biochimica et Biophysica Acta (1976) 455:269-271) who demonstrated that liposomes efficiently concentrated several catecholamines (dopamine, norepinephrine and epinephrine) in response to a transmembrane pH gradient).
The presence of an acidic liposomal interior and a basic to neutral exterior environment allows agents that are primarily in the neutral form at neutral to basic pH and primarily in the charged form at acidic pH to be readily entrapped within a liposome. Drugs containing ionizable moieties such as amine groups are readily encapsulated and retained in liposomes containing an acidic interior. This method, where an ionophore (A23187) is used to generate a pH gradient across a manganese-containing liposome, has been used to load topotecan into cholesterol-free liposomes comprising a PEG-lipid conjugate inserted in the membrane (see WO/0185131). However, successful loading and retention using a transmembrane pH gradient is realized while the internal pH of the liposome is maintained. Since the pH gradient can only be maintained for short periods of time, clinical formulation of drugs into liposomes requires the generation of a pH gradient in liposomes just prior to drug loading. A second disadvantage of this method results from instability of lipid, and some drugs, at acidic pH which prevents the need for long-term storage of the drug loaded liposome. Freezing of liposomal formulations slows the rate of hydrolysis but conventional liposomal formulations often aggregate and leak contents upon thawing unless appropriately selected cryoprotectants are used.
Complexes between drugs such as doxorubicin or ciprofloxacin and divalent metal ions such as Mn2+ have been reported (Bouma, J., et al. (1986) Pharm. Weekbl. Sci. Edn. 16:109-133; Riley, C. M., et al. (1993) J. Pharm. Biomed. Anal. 11:49-59; and, Fenske, D. B. (1998) Biochim. Biophys. Acta. 1414:188-204). Recently, it was reported that uptake of doxorubicin (but not ciprofloxacin) into sphingomyelin/cholesterol LUVs could be carried out with manganese in the internal loading medium without the presence of an ionophore (Cheung et al., Biochimica et Biophysica Acta (1998) 1414:205). It was suggested that a process involving both complex formation between doxorubicin and manganese ions and protonation of doxorubicin inside the liposome resulted in uptake of this particular drug in the presence of manganese ions. Stable entrapment of doxorubicin was reported but this work relied on the use of sphingomyelin/cholesterol liposomes, a formulation noted for optimal drug retention. The methodology reported by Cheung, et al., involving the use of MnSO4 in pH 7.4 HEPES buffer is not reproducible because the metal precipitates from such a buffer.
Various groups have investigated the interaction of metal ions with liposomes with the goal of evaluating the effects of metal cations on vesicle membranes (Steffan et al. (1994) Chem. Phys. Lipids 74(2): 141-150). Divalent metal cations such as Ca2+ have been implicated in the unfavourable formation of metal induced crosslinking of phosphatidylglycerol (PG) containing liposomes due to the negative charge of the liposome surface. Metal ions have also been implicated in increasing the phase transition temperature of negatively modeled membrane systems (Borle, et al., (1985) Chemistry and Physics of Lipids 36: 263-283; Jacobson, et al., (1975) Biochemistry 14(1): 152-161). These studies revealed that the addition of calcium to dipalmitoylphosphatidylglycerol (DPPG) membranes resulted in a phase transition temperature increase by about 50° C. These results indicate that the use of negatively charged lipids in conjunction with metal ions will result in liposomes that exhibit inferior characteristics for in vivo applications.